Treatment including prebiotic composition for use with probiotics

ABSTRACT

A method is described to treat diarrhea, vaginal yeast infections, constipation, heartburn, bloating, indigestion, flatulence or poor nutrient absorption, by administering a formulation of naturally occurring lecithins and/or oleic acid that stimulate the growth and lactic acid producing activity of  Lactobacillus  and  Bifidobacterium  species of probiotic bacteria, and replace polysorbate 80 for those products, and a formulation of  Lactobacillus  and  Bifidobacterium  species of probiotic bacteria. The formulations are in can a stable powdered form.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional No. 61/363,172,filed Jul. 9, 2010.

BACKGROUND

Prebiotics, which increase the in vivo growth rate or activity ofprobiotic bacteria like Lactobacillus and/or Bifidobacterium, aregenerally soluble fiber sources. Probiotics with or without prebioticsare fed to humans or animals to support intestinal health. Soluble fiberprebiotics are not digested by host animal digestive enzymes but rather,are the energy source for the probiotic species and are digested byenzymes produced by the probiotic species. Soluble fiber prebioticsenhance probiotic growth but are not required for growth.

There are some compounds that are semi-strict requirements forLactobacillus and Bifidobacterium growth but that are not required forhuman or animal nutrition and which can serve as prebiotics. One suchcompound is polysorbate 80 that is contained in Lactobacilli MRS Broth(Difco Laboratories, Detroit, Mich.). When Lactobacilli or certainstrains of Bifidobacteria are inoculated into this broth (containing0.1% polysorbate 80) prolific growth results; whereas, in the absence ofpolysorbate 80, growth can be several logarithms lower. The inability ofmany individuals to experience the benefits of probiotic supplements mayrelate to the inability of probiotic strains to grow and colonizeeffectively in the absence of polysorbate 80.

The scientific literature reports that the oleic acid moieties inpolysorbate 80 may provide its stimulating effect (See: Williams, W. L.et al, 1947, J. Biol. Chem., 170, 619-630). However, most probioticstrains are sensitive to the concentration of oleic acid in their growthmedia and respond negatively by producing less lactic acid (which isvital to obtaining strong probiotic benefits) when slight excesses ofoleic acid are present. But these same strains do not respond negativelywhen excess polysorbate 80 is present. So it appears there is somethingunique to the polysorbate 80 stimulation effect, which oleic acid alonecannot duplicate.

The problems with using polysorbate 80 in commercial probiotic productswhen attempting to utilize its dramatic growth enhancing benefitsare: 1) it is not a natural compound and is rejected by those consumersinsisting on natural, organic ingredients; and 2) it reacts with anddegrades freeze-dried probiotic strains when mixed directly with them.So, there is a need for an alternative natural prebiotic that canreplace polysorbate 80 in commercial probiotic formulations, without thedrawbacks mentioned above.

SUMMARY

Naturally occurring lecithins and/or oleic acid can stimulate the growthand lactic acid producing activity of Lactobacillus and Bifidobacteriumspecies of probiotic bacteria, and replace polysorbate 80 as an additivefor those products. They can be administered as either a stable powderedform of these naturally oily compounds (this process also reduces theirchemical reactivity) or in their natural form.

This stable powder form of lecithins and/or oleic acid can be addeddirectly to probiotic formulations containing sensitive, freeze-driedLactobacillus and Bifidobacterium probiotic strains and enhance thegrowth (as measured by CFU (colony forming units)) and also enhance LAPA(lactic acid producing activity). These stable powders can also beseparated from the probiotic formulations, and packaged and sold asseparate dietary supplements, e.g., in capsule or tablet form, to beused with the probiotic formulation as desired—preferably in a formwhere they will be able to combine and assist the probiotic growth andactivity once they reach the intestine. This is accomplished byincorporating them in a capsule or delivery mode that has an entericcoating.

Naturally occurring lecithins and/or oleic acid can also be administeredseparately from the probiotic formulation. Again, they should preferablybe in a form where they will be able to combine and assist the probioticgrowth and activity once they reach the intestine—e.g., by incorporatingthem in a capsule or delivery mode that has an enteric coating.

For the stable powder form of the lecithins and/or oleic acid which areto be separately administered, they are first adsorbed to an adsorbentcompound or composition, including calcium silicate, silicon dioxide,fumed silica, food grade clay substances such as kaolin and sodiumbentonite, food starches such as Pure Dent B830 corn starch (GrainProcessing Corp., Muscatine, Iowa), Perfectamyl D6 potato starch(National Starch-Avebe, Bridgewater, N.J.), and preferably to Hubersorb600 (J.M. Huber Corp., Havre de Grace, Md.), a highly adsorbent form ofcalcium silicate. The formulation also preferably includes dry forms ofmicrocrystalline cellulose and silica. The carrier is preferably 10-300%(by weight) of the adsorbent, and the silica is preferably 0.25 to 2% byweight of the final formulation, with the lecithin/oleic acid preferablyat 5-40% of the final formulation weight.

For a formulation of the naturally occurring lecithins and/or oleicacid, the ratio of lecithin to oleic acid can preferably be from 95:5 byweight to 60:40, with a ratio of 90:10 more preferred. The lecithin canbe any form of lecithin, preferably sunflower lecithin or soy lecithin,and more preferably sunflower lecithin. The preferred embodiment is90:10, sunflower lecithin: oleic acid, with an antioxidant, for example,natural Vitamin E (d-alpha tocopherol), at 0.01% to 5.0%, with 0.1% byweight preferred, of the final formulation.

The dry prebiotic formulation can be incorporated into a final product,including probiotics, at between 1 and 100% of the final productformulation, with 10-20% preferred. Such final product is preferablytaken with a carbohydrate snack of about 50 g in weight, making thelecithin/oleic acid at about 0.01%-5.0% of the total weight of theproducts ingested, with 0.1% by weight being preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the enhanced growth, and FIG. 1B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in the product Theralac®, (Master Supplements, Inc., (Victoria,Minn.)), which primarily consist of Lactobacillus and Bifidobacteriumstrains, with some initial dry-form Lactostim® (Master Supplements,Inc., (Victoria, Minn.)) present.

FIG. 2A shows the enhanced growth, and FIG. 2B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in Udo's Choice Super 8 Hi Potency™, which primarily consist ofLactobacillus and Bifidobacterium strains.

FIG. 3A shows the enhanced growth, and FIG. 3B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in Ultimate Flora™ from Renew Life, which consists ofLactobacillus and Bifidobacterium strains.

FIG. 4A shows the enhanced growth, and FIG. 4B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in Jarrow-Dolphius EPS by Jarrow Formulas, which primarilyconsist of Lactobacillus and Bifidobacterium strains.

FIG. 5A shows the enhanced growth, and FIG. 5B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in PB8 by Nutrition Now, which primarily consist ofLactobacillus and Bifidobacterium strains.

FIG. 6A shows the enhanced growth, and FIG. 6B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in All Flora by New Chapter, which primarily consist ofLactobacillus and Bifidobacterium strains.

FIG. 7A shows the enhanced growth, and FIG. 7B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the probiotic bacterialspecies in Primadophilus Optima by Natures Way, which primarily consistof Lactobacillus and Bifidobacterium strains.

FIG. 8A shows the enhanced growth, and FIG. 8B shows the enhancedactivity, with naturally-occurring sunflower lecithin and oleic acid ata 90:10 weight ratio, including Vitamin E and the Bifidobacterium strainin Align, by Proctor & Gamble.

DETAILED DESCRIPTION

Probiotic formulations are useful in treating a number of conditions,including irritable bowel syndrome, Crohn's disease, remission ofulcerative colitis and preventing relapse of Crohn's disease and therecurrence of pouchitis (following surgery), colitis, ileitis,diverticulitis, re-establishing the intestinal tract's beneficialmicroflora that are destroyed by the antibiotics used to treat diarrhea(including traveler's diarrhea), vaginal yeast infections, constipation,heartburn, bloating, indigestion, flatulence and poor nutrientabsorption. Conditions leading to indigestion and lack of nutrientabsorption by the intestine are also treated by probiotics. The averageperson carries about 4-5 pounds of fecal matter in their intestine, andadding probiotics helps to reduce the volume. With the prebioticformulation herein, the volume of fecal expelled per unit time isgenerally increased, thereby reducing the carried fecal matter.

As prebiotics enhance the efficacy of probiotics, they should be usedwith them when feasible. Probiotics dosing should be at least about30-40 billion CFUs every three days, to as much as 180 billion per day,depending on severity of disease or condition and patient response.There is no known toxic level or excessive dosage. The prebiotics doseto effectively support each probiotic dosage is about 10 to 100 mg ofthe active ingredient(s)—lecithin and/or oleic acid—more preferably,about 20-50 mg of the active ingredients, for each probiotics dose. Ifmore probiotic is taken, the prebiotic can be similarly increased formaximum effect.

The prebiotics of this invention can be in the same capsule orformulation as the probiotics, or in a separate dosage form. Theprebiotics should be in a separate dosage form if they are naturallyoccurring, as opposed to adsorbed. The prebiotics of this invention maybe taken with carbohydrate or fiber to increase their effectiveness.

Prebiotic formulations are often probiotic strain dependent. SomeLactobacillus strains, for example, respond best to a form of lecithinderived from sunflowers (Sunflower Lecithin) while others show nopreference to lecithin type and respond equally well to soy lecithin.Other strains show a synergistic response when lecithin is used togetherwith oleic acid. Verification of the properties of the formulation wasmade by examining the growth (measured in NTU turbidity units) andlactic acid producing activity (measured by titration and pH) in MRSbroth made up such that polysorbate 80 could either be retained (ascontrol) or be removed and substituted by a preferred lecithin and oleicacid mixture, which is 90% sunflower lecithin and 10% oleic acid byweight, and includes 0.1% by weight Vitamin E.

The growth and activity results shown in FIGS. 1A to 8B were with thecommercial probiotic products indicated in the figures, using the liquidform of sunflower lecithin and oleic acid (90:10 weight ratio andincluding 0.1% by weight Vitamin E) or not, as indicated. The growthmedia was the MRS media prepared as described in Example 3 below, andthe testing of growth and activity was as described in Example 4 below.

Example 1 Preparing Prebiotic Formulations Including One or More ofSunflower Lecithin, Soy Lecithin and Oleic Acid

The active ingredients in the formulation: sunflower lecithin, soylecithin and oleic acid, are oily, non-aqueous liquids that should berendered into stable, non-reactive powders to render them suitable forcombination with probiotics in the same formulation by, for example, thefollowing process.

Step 1: Combining Active Ingredients

The active ingredients (e.g., sunflower lecithin, soy lecithin and/oroleic acid) are mixed into an adsorbent material under non-aqueousconditions. The adsorbent is selected from a large class of substancesincluding but not limited to: calcium silicate, silicon dioxide, fumedsilica, food grade clay substances such as kaolin and sodium bentonite,food starches such as Pure Dent B830 corn starch (Grain ProcessingCorp., Muscatine, Iowa) and Perfectamyl D6 potato starch (NationalStarch-Avebe, Bridgewater, N.J.). One preferred substance is Hubersorb600 (J.M. Huber Corp., Havre de Grace, Md.), a highly adsorbent form ofcalcium silicate.

The adsorbent is first placed in a suitable mixer such as a Hobartfitted with either a wire wisp or a pizza dough mixing blade and, withthe mixer running at medium speed (40-60 RPM). The active ingredient isadded slowly by pouring or pumping onto the adsorbent. It is importantto modulate the rate of addition such that the adsorbent is notoverloaded to where lumps form.

The active ingredient(s) should be homogeneously premixed with any othernecessary or desired ingredients (for example, Vitamin E; including anyof the D, L or a mix of the D and L isomers) prior to adding to theadsorbent if such a mixture is required. The Vitamin E is preferably0.05 to 2% of the active ingredients, by weight. The active ingredientsare preferably mixed with Vitamin E separately, and this mixture is thenadded to the adsorbent in the Hobart under constant agitation. For otherproportions, if Hubersorb 600 is selected as the adsorbent for thevitamin E treated sunflower lecithin, active ingredient can be up toabout 150% of the weight of the Hubersorb 600. If Perfectamyl D6 potatostarch is used instead as the adsorbent, then active ingredient addedmay be only about 12% of its weight. A trial an error process isrequired when trying new adsorbent materials from the groups listed.Once all active ingredient(s) have been added, one proceeds to Step 2.

Step 2: Adding Additional Carrier

Once all the active ingredient(s) have been adsorbed, a second carrieringredient selected from a different class is added to the mixture inthe Hobart. This ingredient attenuates the mixture rendering it intosmaller particles. The preferred class of ingredients here are thedriest forms of microcrystalline cellulose available; i.e.,pharmaceutical grades such as Microcel MC-132 (Blanver, Boca Raton,Fla.) or the Avicell PH112 brand (FMC Corporation, Philadelphia, Pa.).The amount added will vary between 10% to 300% of the weight of theadsorbent used. Specific amounts are specified in the examples thatfollow. The microcrystalline cellulose ingredient can be added rapidlywith constant mixing at the highest workable speed, i.e., one that doesnot produce spillage or excessive dusting. Mixing times run 2-10 minutesor until uniform consistency is achieved. This step, as well as Step 1,is best performed in a humidity controlled room where the relativehumidity is 40% or lower, preferably below 25%.

Step 3: Adding Silica

This final step provides for additional drying and producing enhancedflowability of the formula by adding special grades of silica (SiO2). Itcan be done in the same mixer as steps 1 and 2: 0.25-2% by weight of theStep 2 mixture is added as silica, the Syloid 63 brand (W.R. Grace,Columbia, Md.) and the Pirosil brand (AIC, Inc., Framingham, Mass.) arepreferred. The silica is added under constant mixing and mixingcontinues for 2-3 minutes or until uniformity is achieved. All steps 1-3are to be conducted under Food Grade GMP procedures.

For enhanced drying, the silica can be heated in an oven at 450° F. for8 hours and cooled prior to use. This is recommended when this productis added to formulations containing probiotic strains that havesignificant sensitivity to water activity.

Step 4: Packaging

The final formulation is packaged in airtight drums or bags and storedin a cool dry place until packaged or further processed. The shelf life,depending on the formula, will vary from 12-48 months. The percentactive ingredient(s) in the final formulation will typically run from 5%to 40% depending on the formula. A sunflower lecithin formula usingHubersorb 600 as the adsorbent can contain up to 35% active ingredient(sunflower lecithin+vitamin E).

Final formulations from step 3 can be incorporated directly intoprobiotic formulations destined to be filled into capsules or sold aspowders. The amount required to achieve a probiotic stimulation effectgenerally runs from 10 to 100 mg as active ingredient per probioticdose, but more can be used. With a product that contains 30% activeingredient, a 10 mg active ingredient dose is achieved using about 33 mgof product for each probiotic dose in the probiotic formulation.

These results with various formulations made according to the abovesteps are provided in examples 4 to 10 below.

Example 2 Probiotic Cultures

The following probiotic cultures were obtained from Danisco in Madison,Wis. and were used in testing the prebiotic formulations:

Lactobacillus acidophilus NCFM (also called LA-1)

Lactobacillus rhamnosus Lr-32 (also called LR-44)

Lactobacillus paracasei Lpc-37 (also called F-19)

Lactobacillus salavarius Ls-33 (also called LS-30)

Bifidobacterium lactis Bl-04 (also called BL-34)

Once a particular substitute (“active”) ingredient for polysorbate 80(i.e., sunflower lecithin, soy lecithin and/or oleic acid) is determinedfor a given probiotic strain by obtaining an equivalent or close toequivalent measure of growth and activity for that ingredient(s), it isformulated as in Example 1 to render it stable and non-reactive prior toingestion and/or blending it into a commercial probiotic formulation.“Non-reactive” as used herein means that said rendered ingredient willnot destabilize a finished probiotic formulation with respect tooxidation, redox potential, hygroscopicity, water activity, cellmembrane integrity, and pH, or otherwise negatively affect probioticactivity.

Example 3 MRS Broth

MRS broth as used for testing of the formulation herein is made asfollows, where the ingredients include:

Proteose Peptone #3: 10.0 g

Beef Extract: 10.0 g

Yeast Extract: 5.0 g

Glucose: 20.0 g

Polysorbate 80: 1.0 g (or substitute ingredient; or none for control)

Ammonium Citrate: 2.0 g

Sodium Acetate: 5.0 g

Magnesium sulfate: 0.1 g

Manganese sulfate: 0.05 g

Dipotassium phosphate: 2.0 g

Distilled Water: 1 liter

The MRS broth was prepared in 1 liter screw cap Erlenmeyer flasks, asbelow in Example 4, for testing of various probiotic strains for growthand activity with just MRS broth (control), broth plus sunflowerlecithin and/or oleic acid, and broth plus polysorbate 80.

Example 4 Testing Lecithins and/or Oleic Acid as Substitutes forPolysorbate 80 in the Growth of Probiotic Bacteria in MRS Broth

MRS broth was prepared with polysorbate 80 (for controls) and withoutpolysorbate 80 (for tests), and without polysorbate and with Lecithinsand/or oleic acid instead (0.1% by weight of the broth), and filled into1 liter screw cap flasks in equal amounts, which where were thenautoclaved at 121° C. for 15 minutes. Each flask was tempered to 37° C.and aseptically inoculated with 0.14 gm of the individual probioticstrains (in Examples 5 to 10 below) to be tested. At specified timeintervals, a 30 ml sample from each flask was aseptically transferredinto a HACH 2100N Turbidimeter sample cell. The turbidity of each samplewas read and the results were reported in NTU's.

The same 30 ml sample used for the turbidity reading was transferredinto a 250 ml glass beaker, and the pH was recorded. The sample wastitrated using 0.1 N NaOH to an endpoint of pH 6.8, and the quantity ofNaOH used was recorded. The % Lactic Acid was calculated using thefollowing formula:% Lactic Acid=((mls of 0.1 N NaOH)×(0.1 N)×(90 gm/mole)×(1 L/1000ml))×100 mls of sample% Lactic Acid=(mls of 0.1 N NaOH)×(0.9)

The results are presented in Exs. 5-10 below, and in the FIGS. 1A to 8B:

Example 5 Testing L. acidophilus NCFM (LA-1)

(Results reported at 12 hours and 23 hours like: 12/23 in Table I below)

TABLE I NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 1855/2038 1.09/1.27 4.2/4.1 80 in MRS None,MRS only 37/68 0.36/0.49 5.2/4.9  0.1% Sunflower Lecithin 2015/22171.17/1.52 4.1/3.9  0.1% Soy Lecithin 1962/2065 1.20/1.53 4.1/3.9  0.1%Oleic Acid 1451/1789 1.03/1.21 4.3/4.1  0.1% Sunflower Lecithin +2228/2877 1.19/1.57 4.1/4.0   0.1% Oleic Acid  0.05% Sunflower Lecithin 895/1076 0.98/1.33 4.2/4.0 0.025% Sunflower Lecithin 317/376 0.78/1.044.4/4.2 0.025% Oleic Acid 498/603 0.70/0.77 4.5/4.4 0.025% SunflowerLecithin + 2273/2523 1.06/1.47 4.2/3.9 0.025% Oleic Acid  0.1% SunflowerLecithin + 2183/3193 1.16/1.53 4.1/3.9  0.05% Oleic Acid 0.98/1.33 0.05% Sunflower Lecithin + 2330/2701 1.16/1.54 4.1/3.9  0.05% OleicAcid

Example 5 Conclusions

Growth in MRS containing polysorbate 80 is the standard for comparison.Growth in MRS without either polysorbate 80 or one of the test additivesis negligible, indicating the importance of such additives on probioticgrowth. Both lecithins effectively replace polysorbate 80 at the 0.1%inclusion rates. The 0.05% inclusion rate for sunflower lecithin is atthe lowest level of acceptable effectiveness; 0.025% is too low and doesnot yield acceptable stimulation. Oleic acid and sunflower lecithin aresynergistic at the 0.025% inclusions levels, as the combined result issignificantly greater than what was obtained by either additive alone at0.025%. Oleic acid at 0.1% is somewhat effective as a polysorbate 80replacement but not as effective as either sunflower or soy lecithin.

Example 6 Testing Bifidobacterium lactis Bl-04 (BL-34)

(Results reported at 12 hours and 23 hours like: 12/23 in Table IIbelow)

TABLE II NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 80  922/1434 0.57/0.84 4.8/4.5 in MRS None,MRS only 20/32 0.15/0.23 5.4/5.3  0.1% Sunflower Lecithin 1014/1532 0.6/0.92 4.8/4.5  0.1% Soy Lecithin  868/1411 0.59/0.91 4.9/4.7  0.1%Sunflower Lecithin + 1259/1979 0.66/0.99 4.8/4.5 0.25% Oleic Acid

Example 6 Conclusions

Growth in MRS containing polysorbate 80 is the standard for comparison.Growth in MRS without either polysorbate 80 or one of the test additivesis negligible, indicating the importance of such additives for probioticgrowth. Both lecithins (soy and sunflower) effectively replacepolysorbate 80 at the 0.1% inclusion rates but sunflower lecithin givesthe better result. Oleic acid and sunflower lecithin work at the 0.025%inclusion level for oleic acid and at 0.1% for sunflower lecithin,yielding a significant increase in turbidity (cell growth) and a slightincrease in lactic acid production to sunflower lecithin alone.

Example 7 Testing Lactobacillus paracasei Lpc-37 (F-19)

(Results reported at 12 hours and 23 hours like: 12/23 in Table IIIbelow)

TABLE III NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 80 1664/2824 0.99/1.39 4.2/3.9 in MRS None,MRS only 21/43 0.26/0.36 5.4/5.1 0.1% Sunflower Lecithin 1717/28130.91/1.34 4.2/3.9 0.1% Oleic Acid  178/1789 0.33/1.01 5.1/4.1 0.1%Sunflower Lecithin +  335/2103 0.29/1.00 5.3/4.1 0.1% OleicAcid

Example 7 Conclusions

Growth in MRS containing polysorbate 80 is the standard for comparison.Growth in MRS without either polysorbate 80 or one of the test additivesis negligible, indicating the importance of such additives for probioticgrowth. Sunflower lecithin effectively replaces polysorbate 80 at the0.1% inclusion rate. Oleic acid at 0.1% shows an improvement over nopolysorbate 80 but is not as effective a replacement for polysorbate 80as sunflower lecithin. Oleic acid does not appear to be synergistic withsunflower lecithin at the concentrations studied.

Example 8 Testing Lactobacillus rhamnosus—Lr-32 (LR-44)

(Results reported at 12 hours and 23 hours like: 12/23 in Table IVbelow)

TABLE IV NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 80 2804/3776 1.12/1.67 4.0/3.7 in MRS None,MRS only 40/72 0.46/0.52 5.0/4.8 0.1% Sunflower Lecithin 2572/36571.07/1.71 4.1/3.7 0.1% Oleic Acid −30.9/2644  0.072/1.05  6.2/4.1 0.1%Sunflower Lecithin +  57/3605 0.13/1.25 6.0/3.9 0.1% OleicAcid

Example 8 Conclusions

Growth in MRS containing polysorbate 80 is the standard for comparison.Growth in MRS without either polysorbate 80 or one of the test additivesis negligible, indicating the importance of such additives for probioticgrowth. Sunflower lecithin effectively replaces polysorbate 80 at the0.1% inclusion rate. Oleic acid at 0.1% shows an improvement over nopolysorbate 80 on the 23-hour test but shows a significant lag phase at12 hours; it is not as good a replacement for polysorbate 80 assunflower lecithin. Oleic acid is not synergistic with sunflowerlecithin at the concentrations studied.

Example 9 Testing Lactobacillus salivarius—Ls-33 (LS-30)

(Results reported at 12 hours and 23 hours like: 12/23 in Table V below)

TABLE V NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 80 3426/3931 1.42/1.68 3.8/3.7 in MRS 0.1%Sunflower Lecithin 3040/3283 1.36/1.60 3.9/3.8 0.1% Oleic Acid 1440/35780.073/1.51  4.4/3.8

Example 9 Conclusions

Sunflower lecithin effectively replaces polysorbate 80 at the 0.1%inclusion rate. Oleic acid at 0.1% is not as good a replacement as a lagphase is apparent at 12 hours.

Example 10 Growth of the Commercial Probiotic THERALAC® in MRS BrothContaining Polysorbate 80, Compared to Oleic Acid and Lecithin

MRS broth was prepared with (for controls) and without (for tests)polysorbate 80 and filled into 1 liter screw cap flasks at 500 ml eachand autoclaved at 121° C. for 30 minutes. Various lecithin and/or oleicacid additives, or nothing (negative control), was added to the testsflasks prior to autoclaving. The flasks were then tempered to 37° C. ina water bath and inoculated at 0.1% with THERALAC® Lot #32310A, acommercial probiotic product (Master Supplements, Inc., Victoria,Minn.). Capsules of THERALAC® were opened and the contents mixedaseptically to serve as the inoculum source. At an inoculation rate of0.1%, approximately 80 million CFU was added per ml of MRS media.THERALAC® contains (formulation strength) the following probioticstrains at the approximate CFU values listed per gram:

Lactobacillus acidophilus NCFM (LA-1): 30 billion CFU

Bifidobacterium lactis Bl-04 (BL-34): 30 billion CFU

Lactobacillus paracasei Lpc-37 (F-19): 14 billion CFU

Lactobacillus rhamnosus Lr-32 (LR-44): 6 billion CFU

Bifidobacterium lactis Bi-07: 8 billion CFU

Turbidity, pH and titrations (made with 0.1 N NaOH and reported as %lactic acid) were made at various times up to 23 hours. The results arepresented below in Table VI for the 12 and 23-hour analyses.

TABLE VI (Results reported at 12 hours and 23 hours like: 12/23 in TableVI below) NTU Lecithin/Oleic Acid in MRS (Turbidity) Lactic Acid % pHNone: included Polysorbate 80 3311/3694 1.42/1.84 3.9/3.7 in MRS None,MRS only 44/75 0.44/0.56 5.0/4.8 0.1% Sunflower Lecithin 3343/37581.45/1.82 3.9/3.7 0.1% Soy Lecithin 2939/3563 1.33/1.84 4.0/3.7 0.1%Oleic Acid 2775/3529 1.18/1.72 4.1/3.8 0.1% Sunflower Lecithin +2488/2917 1.20/1.72 4.0/3.8 0.1% OleicAcid 0.1% Sunflower Lecithin +3447/3724 1.40/1.80 3.9/3.8 0.025% Oleic Acid 0.1% Soy Lecithin +3190/3607 1.42/1.80 3.9/3.8 0.025% OleicAcid

Example 10 (THERALAC® TEST) Conclusions

Growth in MRS containing polysorbate 80 is the standard for comparison.Growth in MRS without either polysorbate 80 or one of the test additivesis negligible, indicating the importance of such additives for probioticgrowth. Both lecithins effectively replace polysorbate 80 at the 0.1%inclusion rates but sunflower lecithin shows significantly more cellgrowth (turbidity) and lactic acid production at 12 hours, indicating areduced lag phase. Oleic acid alone at 0.1% shows acceptable replacementvalue for polysorbate 80 but is not as effective as either of thelecithins. There appears to be a slight synergism between soy lecithinand oleic acid at the concentrations studied.

Example 11 Preparation of a Dry, Stable, Non-Reactive Prebiotic (DSNRP#1) for Use In Commercial Probiotic Formulations

Step A: Add 250 grams of Hubersorb 600 to the bowl of a Kitchen Aid™orbital mixer utilizing the wire wisp attachment.

Step B: In a separate container add 0.38 grams of natural vitamin E oil(Sigma Aldrich, St. Louis, Mo.) to 320 grams of sunflower lecithin(GIRALEC—Austrade, Inc., Palm Beach Gardens, Fla.) and 60 grams ofextra-virgin olive oil. Mix thoroughly.

Step C: Start the agitation in the Kitchen Aid™ mixer at medium speed(about 40-50 RPM) and slowly pour in (add at approx. 100 ml/min.) the380 grams of vitamin E treated sunflower lecithin plus olive oil. Mixfor 2-3 minutes at medium-high speed (approximately 50-70 RPM) until theliquid is completely adsorbed.Step C: While continuing to mix slowly add 560 grams of Microcel MC-132microcrystalline cellulose (Blanver, Boca Raton, Fla.) and continue tomix for another 1-2 minutes.Step D: Add 22 grams of Pirosil™ silica (AIC, Inc., Framingham Mass.) tothe mixture and continue to mix for 2 additional minutes.

The final product is a free flowing white powder containing 26.4%sunflower lecithin by weight. DSNRP #1 for dry probiotic formulations(to be filled into capsules) would be 10% by weight of the formulation,where the probiotic dose rate is two 500 mg capsules per day. Thisprovides 100 mg of DSNRP #1 containing 26 mg of active ingredient(sunflower lecithin). When these two capsules are taken with a 50 gramcarbohydrate snack, the resulting concentration of lecithin will be0.05%—an effective amount according to the results indicated herein.

Example 12 Preparation of a Dry, Stable, Non-Reactive Prebiotic (DSNRP#2) for Use In Commercial Probiotic Formulations

Step A: Add 250 grams of Hubersorb 600 to the bowl of a Kitchen Aid™orbital mixer utilizing the wire wisp attachment.

Step B: In a separate container add 0.38 grams of natural vitamin E oil(Sigma Aldrich, St. Louis, Mo.) to 320 grams of sunflower lecithin(GIRALEC—Austrade, Inc., Palm Beach Gardens, Fla.) and 60 grams of oleicacid (Sigma-Aldrich). Mix thoroughly.

Step C: Start the agitation in the Kitchen Aid™ mixer at medium speed(about 40-50 RPM) and slowly pour in (add at approx. 100 ml/min.) the380 grams of vitamin E treated sunflower lecithin plus oleic acid. Mixfor 2-3 minutes at medium-high speed (approximately 50-70 RPM) until theliquid is completely adsorbed.Step C: While continuing to mix slowly add 560 grams of Microcel MC-132microcrystalline cellulose (Blanver, Boca Raton, Fla.) and continue tomix for another 1-2 minutes.Step D: Add 22 grams of Pirosil™ silica (AIC, Inc., Framingham, Mass.)to the mixture and continue to mix for 2 additional minutes.

The final product is a free flowing white powder containing 26.4%sunflower lecithin and 5% oleic acid by weight. DSNRP #2 in dryprobiotic formulations (to be filled into capsules) would be preferably10% by weight of the formulation, where the probiotic dose rate is two500 mg capsules per day. This provides 100 mg of DSNRP #2 containing 26mg of sunflower lecithin and 5 mg of oleic acid). When these twocapsules are taken with a 50 gram carbohydrate snack, the resultingconcentration of the combined active ingredients will be 0.062%—aneffective amount according to the results indicated herein.

Example 13 Preparation of a Dry, Stable, Non-Reactive Prebiotic (DSNRP#3) for Use In Commercial Probiotic Formulations

Step A: Add 780 grams of Perfectamyl D6 potato starch (NationalStarch-Avebe, Bridgewater, N.J.) to the bowl of a Kitchen Aid™ orbitalmixer utilizing the wisp attachment for agitation.

Step B: In a separate container add 0.12 grams of natural vitamin E oil(Sigma-Aldrich, St. Louis, Mo.) to 100 grams of soy lecithin (ADM, Inc.,Decatur, Ill.) and 20 grams of extra-virgin olive oil. Mix thoroughly.

Step C: Start the agitation in the Kitchen Aid™ mixer at about 40-50 RPMand slowly pour in (add at approx. 100 ml/min.) the 120 grams of liquidfrom step B. Mix for 3-4 minutes until completely adsorbed.

Step C: While continuing to mix slowly add 80 grams of Avicel PH112brand (FMC Corp., Philadelphia, Pa.) microcrystalline cellulose andcontinue to mix for another 2-3 minutes.

Step D: Add 20 grams of Syloid 63 silica (W.R. Grace, Inc., Columbia,Md.) to the mixture and continue to mix for 3 additional minutes.

The final product is a free flowing white powder containing 10% activeprebiotic ingredient (soy lecithin) by weight. DSNRP #3 for dryprobiotic formulations (to be filled into capsules) would be 20% byweight of the formulation where the probiotic dose rate is two 500 mgcapsules per day. This provides 200 mg of DSNRP #3 containing 20 mg ofactive ingredient (soy lecithin). When such a probiotic is taken with a20 gram carbohydrate snack the resulting concentration of prebiotic willbe 0.1%—an effective amount according to the results indicated herein.

Example 14 Preparation of a Dry, Stable, Non-Reactive Prebiotic (DSNRP#4) that Can be Added to Commercial Probiotic Formulations

Step A: Add 780 grams of food grade Kaolin clay (Vanclay, R.T.Vanderbilt Co., Norwalk, Conn.) in the bowl of a Kitchen Aid™ orbitalmixer utilizing the pizza dough mixing impeller.

Step B: In a beaker add 0.12 grams of natural vitamin E oil (SigmaAldrich, St. Louis, Mo.) to 100 grams of sunflower lecithin and 20 gramsof olive oil. Mix thoroughly.

Step C: Start the agitation in the Kitchen Aid™ mixer at about 40-50 RPMand slowly pour in (add at approx. 100 ml/min.) the 120 grams of vitaminE treated sunflower lecithin plus olive oil. Mix for 3-4 minutes.

Step C: While continuing to mix slowly add 80 grams of Microcel MC-132microcrystalline cellulose and continue to mix for another 2-3 minutes.

Step D: Add 20 grams of Pirosil silica to the mixture and continue tomix 3 additional minutes.

The final product is a free flowing beige-colored powder containing 10%active prebiotic ingredient (sunflower lecithin) by weight. DSNRP #4 fordry probiotic formulations (to be filled into capsules) would be 20% byweight of the formulation, where the probiotic dose rate is two 500 mgcapsules per day. This provides 200 mg of DSNRP #4 containing 20 mg ofactive ingredient (sunflower lecithin). When such a probiotic is takenwith a 20 gram carbohydrate snack the resulting concentration ofprebiotic will be 0.1%—an effective amount according to the resultsindicated herein.

Example 15 Direct Dietary Supplement Use of DSNRP Prebiotic Formulations

The DSNRP formulations from examples 11-14 can be filled into hardcapsules such as gelatin or HPMC capsules, or any other capsule typessuitable for use with dietary supplements, in such sizes as 50 mg to1,000 mg, and utilized for direct consumption as a dietary supplementindependent of any probiotic product, or can be taken with a probioticproduct, or the probiotic product can be included in the formulation. Asnoted above, all such formulations preferably include an entericcoating; for example, the one described in U.S. Pat. Nos. 7,229,818 and7,122,370 (incorporated by reference). Magnesium stearate at 2-3% byweight of the DSNRP formulas should be included to serve as anencapsulating lubricant. Tablets and caplets can also be produced fromthe DSNRP formulations as an option to capsules. Final encapsulated ortableted DSNRP products should be packaged in hermetically sealedplastic or glass bottles with moisture absorbent packets and thefinished products should be stored under cool conditions. The separateformulations can be administered before or after administeringprobiotics, under any of a number of dosing schedules.

The foregoing examples, other terms and expressions, are given for thepurpose of illustrating various embodiments of the invention and, alongwith the methods described herein, are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the claims,will occur to those skilled in the art. The invention is defined only inthe claims which follow and includes all equivalents of the claims.

What is claimed is:
 1. A method of treating diarrhea, vaginal yeastinfections, constipation, heartburn, bloating, indigestion, flatulenceor poor nutrient absorption, comprising: administering a formulation ofa dry powdered form of probiotic bacteria including Lactobacillus orBifidobacterium; administering a formulation of a dry powdered form of aprebiotic including sunflower lecithin or oleic acid, or combinations ofsunflower lecithin and oleic acid, and wherein, the formulationssubstantially free of polysorbate
 80. 2. The method of claim 1, whereinthe prebiotic formulation is adsorbed to one or more of calciumsilicate, silicon dioxide, silica, clays and food starches, and mixedwith 10 microcrystalline cellulose and silica.
 3. The method of claim 1,wherein prebiotic formulation is administered separately from theLactobacillus or Bifidobacterium species.
 4. The method of claim 1,wherein the ratio of sunflower lecithin to oleic acid is 90:10.
 5. Themethod of claim 1, further administering carbohydrates with thesunflower lecithin or oleic acid, or with the combinations of sunflowerlecithin and oleic acid.
 6. The method of claim 1, wherein 10 to 100 mgof the sunflower lecithin or oleic acid, or 10 to 100 mg of thecombinations of sunflower lecithin and oleic acid, are administered foreach dose of the Lactobacillus and/or Bifidobacterium.
 7. The method ofclaim 2, wherein the sunflower lecithin or oleic acid, or thecombinations of sunflower lecithin and oleic acid, are non-aqueous. 8.The method of claim 2, wherein the sunflower lecithin or oleic acid, orthe combinations of sunflower lecithin and oleic acid, are in a capsule,caplet, tablet or other carrier together with Lactobacillus and/orBifidobacterium species.
 9. The method of claim 2, wherein the probioticformulation includes Lactobacillus, Bifidobacterium and other probioticstrains.
 10. The method of claim 2, wherein the food starches are cornstarch or potato starch.
 11. The method of claim 2, wherein the claysare food grade.
 12. The method of claim 2, wherein the clays are kaolinor sodium bentonite.
 13. The method of claim 3, wherein prebioticformulation is administered before or after the probiotic bacteria. 14.The method of claim 3 further including Vitamin E.
 15. The method ofclaim 3, wherein the prebiotic formulation is in a capsule or caplet.16. The method of claim 5, wherein 50 g of carbohydrate are administeredwith the sunflower lecithin or oleic acid, or with the combinations ofsunflower lecithin and oleic acid.
 17. The method of claim 6, wherein 20mg of the sunflower lecithin or oleic acid, or 20 mg of the combinationsof sunflower lecithin and oleic acid, are administered for each dose ofthe Lactobacillus and/or Bifidobacterium.
 18. The method of claim 9,wherein the strains of Lactobacillus include Lactobacillus acidophilusNCFM, Lactobacillus rhamnosus Lr-32, Lactobacillus paracasei Lpc-37, 25and Lactobacillus salavarius Ls-33.
 19. The method of claim 8, whereinthe Bifidobacterium is Bifidobacterium lactis Bl-04 (BL-34).
 20. Themethod of claim 8, wherein the tablet, caplet, capsule or carrier has anenteric coating.
 21. The method of claim 14, wherein Vitamin E is the D,L or a mixture of the D and L isomers.
 22. The method of claim 15,wherein the caplet or capsule has an enteric coating.
 23. The method ofclaim 21, wherein the Vitamin E is 0.05 to 2% by weight of theformulation.